Quick Links

How would you like to share?

Wandering from cell to cell seeding aggregation and wreaking neurodegenerative havoc may not be the image that comes to mind when one thinks of the intracellular protein tau, but a study published in the June 7 Nature Cell Biology hints that that might be exactly what goes on in the Alzheimer brain. Researchers report that from a tiny injected seed, tau pathology eventually spreads to anatomically connected areas of the mouse brain. “It suggests that some form of assembled tau is taken up by cells and then promotes formation and release of new assemblies,” said Michel Goedert, MRC Laboratory of Molecular Biology, Cambridge, UK, one of the senior authors on the paper. “The idea has been out there for a while that normal neuronal transport mechanisms may be causing the spread of tau pathology,” suggested Lary Walker of Emory University, Atlanta, Georgia. Walker has studied protein seeding for some time, but was not involved in this study. “The early Braak studies showed that tau pathology progressed from one region of brain to another, starting in the trans-entorhinal cortex. That made sense, based on connectivity of the regions, but this is the first more or less direct evidence that this might be what’s happening,” Walker told ARF (see also comment below).

Goedert said the study, a collaboration with Markus Tolnay’s group at the University of Basel, Switzerland, was, in fact, inspired by Braak staging of Alzheimer disease (AD). The findings add weight to reports that tau pathology can spread from cell to cell in vitro. Marc Diamond, University of California, San Francisco, found that tau aggregates can get into and corrupt normal tau inside cells and that once aggregation occurs in one cell, it can trigger transfer to adjacent cells in culture (see ARF related news story and ARF news story). “I think our data help support their findings and their findings give our work in vivo relevance,” Diamond said in an interview with ARF.

Diamond also suggested the work might have implications that reach far beyond tau pathology and Alzheimer disease (AD). “One of the reasons this paper is so significant is it comes on the heels of observations on Parkinson’s patients who received neural transplants,” said Diamond. Those studies showed that Lewy bodies, which are predominantly made up of aggregates of the intracellular protein α-synuclein, also developed in the grafted tissue (see ARF related news story). “One interpretation of those observations is that some aggregated protein in the neighboring cells can corrupt the protein in the new cells,” said Diamond. If true, that would support the idea that α-synuclein, tau, and possibly other proteins involved in neurodegeneration, such as huntingtin (see ARF related news story), may behave somewhat like prions.

To test the idea that tau pathology can self-propagate, first author Florence Clavaguera and colleagues took brain samples from P301S tau transgenic mice, which develop filamentous tau aggregates, and injected them into the hippocampus or cerebral cortex of another transgenic strain—the ALZ17 mouse. ALZ17 mice overexpress normal human tau but do not develop tau pathology—though they may be more vulnerable to it. Homogenates from six-month-old P301S mice induced the formation of tau filaments when injected into three-month-old ALZ17 mice. Extracts from non-transgenic mice had no effect. Since human tau expressed in the ALZ17 mice has a different N-terminal sequence to that of the P301S tau, the researchers could ensure that the tau filaments formed were indeed due to the endogenous ALZ17 tau, and not to the injected material. Extracts immuno-depleted of tau had no activity.

The induction of tau filaments was dependent on both time and place. The process was slow. Pathology was first apparent at six months and became more robust at 12–15 months. Filaments appeared faster in the hippocampus than in the cortex. The pathology seems to be caused by some form of insoluble tau, since extracts of soluble material were 20-fold less potent. “Currently, all we know is that there is something in the brain extracts that is quite powerful at inducing tau pathology and that it is insoluble,” said Goedert. “We would like to find out what that substance is, whether it is fibrils, some oligomeric form, or what exactly it is that has the activity.” The same question is plaguing researchers trying to find what entity seeds amyloid-β aggregation in vivo (see ARF related news story and Bolmont et al., 2007).

Interestingly, the pathology spread in the injected mice in a manner that is consistent with the Braak staging idea in that it seems to occur between anatomically connected areas of the brain. But it also appeared in regions that are not typically associated with AD tau pathology. “I am surprised by the number of areas where they see positivity,” said Walker. “It’s everything from the medial lemniscus, which is in a sensory pathway in the brain, to the hypothalamus and the zona incerta. A lot of different areas are affected, but they do have connections with areas that are connected to the injection site,” he said. Goedert stressed that he and his collaborators are not suggesting this as a model for tau transmission in the human brain. “All we can say is that regions where pathology appeared over time were all anatomically connected,” he said.

Interestingly, even though the injected ALZ17 mice developed robust tau pathology, it did not lead to neurodegeneration and cell loss that occurs in the P301S mice. “This suggests that the molecular tau species responsible for transmission and neurotoxicity are not identical,” write the authors. They suggest that, much like prions, distinct tau strains are at work in the mice and may also underlie different tauopathies, such as Pick disease, progressive supranuclear palsy, and AD, which are characterized by different tau isoform assemblies. That’s not to say that tau is a prion. “The big difference is the ease with which the diseases are communicated,” said Walker. “There’s no evidence that tauopathy or Aβ can be transmitted in the same way as prion diseases can, so there may be something about prions that makes them better at transmitting structural information that causes disease.” Diamond suggested that one thing that makes prions unique is that they are incredibly tough. “You can eat them and they will get into your brain. Most of these other proteins, tau, and α-synuclein are vulnerable to proteases and readily digested,” he said.

If tau and α-synuclein toxicities do propagate in some prion-like fashion, then might that offer some new clues as to how to treat these diseases? “That might be possible, but the first thing we have to do is understand the mechanism,” said Goedert. Diamond expressed a similar sentiment. “You can think about disease from the standpoint of what happens in one cell, such as tau gets phosphorylated, misfolds, and causes the cell to get sick, but if you are thinking about how the process propagates between cells, that’s an entirely new way of thinking about therapies,” he said. He suggested that the reach of antibody therapy might extend to these intracellular proteins, for example. “If you understand the mechanisms of how aggregates move between cells and corrupt the protein on the inside of cells, that’s a new therapeutic target that could become a silver bullet for all neurodegenerative diseases that are associated with fibrillar proteins, since we have now seen this prion-like property in tau, α-synuclein, and Aβ,” he said.

Walker agreed. “Mechanistically, the fact that these diseases behave so similarly in tissues and cells, suggests there might be some common mechanism that will help us to understand not just Alzheimer disease but maybe 30 to 40 different proteopathies that all involve the accumulation of protein,” he said.—Tom Fagan

Comments

Comments on News and Primary Papers

The title of this remarkable paper in Nature Cell Biology highlights two key findings: that a tau pathology can be transmitted in vivo, and that it spreads through the brain. The former is a feature of transmissible prions (a characteristic as enigmatic as to elude comprehension), while the latter addresses a key feature of the human Alzheimer pathology, one that despite major recent achievements has not been fully modeled in transgenic mice.

The study uses two mouse strains: ALZ17 mice express high levels of wild-type human tau but reveal only a modest pathology: amyotrophy in the absence of obvious neuronal cell loss and, despite massive hyperphosphorylation of tau, no formation of tau-containing neurofibrillary lesions. In contrast, P301S mice express, at levels comparable to the ALZ17 mice, a mutant form of tau found in familial cases of frontotemporal dementia. Although rarer than the P301L mutation, this mutation causes an earlier onset of pathology in humans. This may explain why the P301S mice present with a particularly robust phenotype, characterized by neurodegeneration in the spinal cord and an abundance of neurofibrillary tangles.

In their study, the research teams lead by Markus Tolnay and Michel Goedert investigated whether aggregation of tau can be transmitted in mice. They intracerebrally injected diluted brain extracts from six-month-old P301 mice into three-month-old ALZ17 mouse and analyzed the injected brains for up to 15 months post-injection. They found that in ALZ17 mice, the P301S extract induced the formation of neurofibrillary tangles as revealed by Gallyas silver impregnation, and reactivity with antibody AT100 that is specific for tau phosphorylated pathologically at the phospho-epitope Thr212/Ser 214.

Interestingly, Clavaguera and colleagues found that Gallyas reactivity (i.e., tangle formation) was not confined to the site of injection, but rather induced up to 2 mm distant of the injection site. A carefully performed time-course analysis suggests a stereotypical mode of spreading (a feature characteristic of Alzheimer disease). It would be interesting to determine how pronounced the pathology of P301S mice would be and how the pathology would spread when injected with a P301S extract, and whether an injection of the P301S extract into the cerebellum (a site spared from pathology) would be sufficient to induce a pathology in this brain area.

Another remarkable finding is that insoluble rather than soluble tau is responsible for the induction of a tau pathology. Here, a follow-up study could determine how long the injected material needs to be around in the brain to exert toxicity and how long it does persist in the mouse brain, questions that could be addressed with biotinylated or fluorescently labeled material.

The paper is a rich source of findings that are worthwhile to be taken up experimentally: Induction of a tau pathology seems to be mediated by oligodendroglia (a cell type not affected by tau pathology in the parental P301S mice). Then, there is evidence for a role of secreted or diffuse tau in propagating tauopathy, rather than of merely cytoplasmic tau. Does this species need to be truncated, specifically phosphorylated or be of a particular higher order assembly? Finally, it seems that there are different species of tau (as in prion diseases) with some species inducing spreading of pathology and others neurodegeneration. As the P301S mice show massive motor neuron loss in spinal cord, it might be worthwhile injecting the P301S extract into brain stem or spinal cord of ALZ17 mice to determine whether in this cellular environment, the tau species present in the P301S extract would induce both, spreading of a ”Gallyas pathology” and degeneration of motor neurons.

In conclusion, this is a true milestone paper that has brought the field forward quite a bit, causing a paradigm shift in tau research. While we are left with many answers there are also a lot of challenging, new questions to be addressed.

Clavaguera and colleagues demonstrate in this elegant study that an injection of a brain extract from P301S tau mice induces aggregation of wild-type tau in mice expressing human or mouse tau. The pathology spreads to anatomically connected brain regions in mice transgenic for human tau but not in wild-type mice expressing only mouse tau. As discussed, tau expression levels may influence the spread, and human tau may also be more prone to aggregate than mouse tau.

These interesting and important in vivo findings tie nicely in with recent cell culture data (1), and support the view that clearance of extracellular tau may have a therapeutic utility, ideally in concert with removal of pathological intracellular tau (2,3). As previously reported, extracellular tau may not only be derived from dead cells but may be secreted and have an extracellular function (4). As discussed in more detail elsewhere (3), it is then conceivable that clearance of extracellular tau can enhance secretion of intracellular tau through a shift in equilibrium, and indirectly reduce pathological tau burden within cells. Furthermore, not only may glia take up extracellular tau but also neurons. This uptake/secretion pathway may be particularly prominent under pathological conditions, and could explain the anatomical spread of tangles during disease progression. All amyloid diseases may be transmissible under certain conditions (5).

The work on murine models of tauopathies conducted by Clavaguera et al. has brought an intriguing view that fibrillar tau pathologies are intracranially transmittable from a single site affected by injected and possibly endogenous tau aggregates. The spreading of Gallyas-positive tau depositions was seemingly consequent to a chain reaction of fibrillogenesis consisting of either transgenically overproduced or endogenously expressed wild-type tau proteins, while the injected brain extracts from transgenic mice expressing the FTDP-17 P301S mutant tau only gave the initial seed for this surge of tangles. Since a PBS-soluble fraction of the extract did not induce overt changes in tau pathology, it is unlikely that monomeric foreign tau proteins convert the conformation of resident tau molecules from a flexible mode to a rigid, more amyloidogenic type, but insoluble tau assemblies preformed in the donor mice acted as seeds of massive inclusions. Pieces of these protein chunks might be axonally transported, and could be the secondary seeds at remote regions. Mechanisms by which alien tau aggregates enter neuronal and glial cells to contact intracellular tau species are so far unknown, but one speculative possibility is that the injectants are endocytosed and attract autophagically sequestered resident tau proteins in endosomes or lysosomes. Such compartmentalization might explain the lack of neurodegenerative alterations in the recipient animals, despite the high abundance of abnormal fibrils. It is yet to be clarified whether such endosomal/lysosomal tau assemblies could grow into neurofibrillary tangles (NFTs) and coiled bodies without structural disruptions of these vesicles.

It should also be noted that all tau molecules seen in this experiment, mutant and wild-type, transgenic and endogenous, were four-repeat tau isoforms (4RTs) bearing exon 10, as only 4RTs are endogenously expressed in adult rodents. Although 4RT filaments could be a seed of exclusive 4RT inclusions via a selective incorporation of 4RT into fibrils, this notion needs to be examined in the same transgenic strain receiving an injection of postmortem brain extracts from tauopathy patients with 4RT-dominant (e.g., progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease), or three-repeat tau isoform- (3RT-) dominant (e.g., Pick disease) pathologies. Alternatively, transgenic mice generated with the use of the genomic human tau DNA, which expresses all six human tau isoforms besides endogenous 4RTs (1,2), could be treated with these extracts to assess whether exogenous insoluble 4RT and 3RT yield their replicas in living brains. Likewise, the formation of NFTs constituted of all six tau isoforms would be experimentally generated by injecting homogenates of Alzheimer disease brains into the genomic tau transgenic mice. An additional assumption to be tested in these analyses is that oligodendrocytes prefer 4RT aggregation to 3RT+4RT fibrils, resulting in the lack of prominent glial tau pathologies as in Alzheimer disease and several other tauopathies with six insolubilized tau isoforms (FTDP-17s with the G272V, V337M, and R406W tau mutations, diffuse neurofibrillary tangles with calcification, etc.).

The 4RT and 3RT aggregates are ultrastructurally identified as straight filaments (SFs) and/or twisted ribbons, and are distinguishable with paired helical filaments (PHFs) characteristic of Alzheimer disease type 3RT+4RT polymers. Moreover, many β-sheet-binding imaging agents show high affinities for Aβ plaques and/or NFTs in Alzheimer disease, but only a small subset of these compounds, as exemplified by X-34 analogs (3-5), are capable of binding to 4RT and 3RT inclusions. This might also support differential accessibilities of SFs and PHFs to intracellular components responsible for tau processing, eventually producing heterogeneous neuropathological phenotypes in tauopathies. X-34 and its derivatives are not optimal for in-vivo positron emission tomographic (PET) imaging, while the development of agents with faster kinetics more suitable for PET assays is ongoing. Longitudinal PET scans with these emerging radiotracers would provide four-dimensional maps of fibrillar tau pathologies spreading in the brain, which might be distinctive depending on the isoform composition of tau deposits. This technology, in conjunction with currently available high-resolution scanners (6), or more advanced future systems, would help researchers and clinicians to spot the site of the primal seeding. If tauopathies are not of multifocal origin, there would be a chance for local injections of genetic, immunological, and pharmacological erasers of the seeds and incipient pathologies. The therapeutic effects and “local recurrence” of the tau deposition after the treatment could also be monitored by the in-vivo imaging techniques.

Clavaguera, Tolnay, Goedert, and colleagues present a compelling argument for the exogenous induction and endogenous spread of tauopathy in rodent models. In these experiments, tauopathy was seeded de novo both in a transgenic mouse strain that normally does not generate filamentous tau, and even (to a lesser degree) in non-transgenic mice. Insoluble tau was the most potent seed, and in both murine host strains the tau filaments that developed consisted of host tau protein. Three key findings are that 1) tauopathy can be seeded within neurons in the living brain by an exogenous seed; 2) once initiated, tauopathy spreads from one brain region to another, possibly via a chain reaction of molecular corruption along with intracellular and intercellular trafficking; and 3) aggregated tau (like prions, Aβ, and probably other pathogenic proteins) may exist as polymorphic and polyfunctional strains, the pathogenicity of which is governed by the characteristics of the corruptive seed and of the host. The findings add to the evidence that disorders of protein aggregation may originate and amplify by a similar molecular mechanism, i.e., by the seeded corruption of endogenously produced molecules. While the various proteopathies may not be communicable in exactly the same sense as are the prionoses, the mechanistic similarities can be instructive, and argue yet again for a more integrative dialogue among researchers studying a wide spectrum of deceptively dissimilar diseases.

Propagation and Prion-like Spreading of Proteins in Common Neurodegenerative Disorders; New Perspectives Emerging From Tau and Synuclein
Many major neurodegenerative diseases are characterized by protein aggregation and deposition in specific regions of brain. This protein pathology generally occurs in discrete regions of brain but eventually spreads into much larger areas (1,2). Several recent studies propose a prion-like, templated aggregation hypothesis regarding the mechanism underlying this propagation of disease-specific protein aggregation (3-5). The most recent report supporting this hypothesis has come from the work by Goedert, Tolnay, and their colleagues, who studied the propagation of tauopathy in transgenic mouse brain (6). In this study, they injected the brain extract of P301S tau transgenic mouse, which has filamentous tau aggregates, into the hippocampus and cerebral cortex of ALZ17, a transgenic line overexpressing wild type tau protein, and examined the spread of tau pathology over time. They found the spread of tauopathy not only within the injection sites but also in neighboring brain regions, with the severity diminishing as the distance from the injection site increases. Both neuropil threads and neurofibrillary tangles were found, as well as oligodendroglial coiled bodies, and the lesions increased with time up to 15 months post injection. When the P301S brain extract was injected to non-transgenic mouse, the aggregation of mouse endogenous tau was induced, but this mouse tau aggregation was confined near the injection site and temporal progression was not observed between six and 12 months. Whether this is due to the species difference or related more to expression levels remains to be determined.

This prion-like spread of protein pathology has also been shown for amyloid-beta aggregates in a transgenic mouse model (5). However, the tau study is particularly striking as this protein and its aggregates have been believed to reside within the cytoplasm. Earlier this year, tissue culture studies with mutant huntingtin fragments and tau proposed aggregate spreading through cell-to-cell transmission of misfolded proteins (7,8). More recently, our laboratories demonstrated the propagation of a-synuclein aggregates through cell-to-cell transmission in cultured cells and transgenic mouse models (9). This transmission apparently involves the release of a-synuclein aggregates from neuronal cells under stresses and subsequent endocytosis by neighboring cells. This interneuronal transmission of a-synuclein may account for the recent reports showing the spread of Lewy inclusions from host tissues to long-term fetal cell grafts in Parkinson’s patients and may be the underlying mechanism for the sequential progression of the brain stem-originated Lewy pathology in PD, proposed by Braak and colleagues. Therefore, this prion-like propagation of pathological aggregation may be the general underlying principle for the progressive deterioration of myriad neurodegenerative diseases associated with protein misfolding.

There are several outstanding questions. First, given the cytosolic localization of tau and other disease-linked proteins, mechanisms must exist by which these proteins are released from neurons and gain access to the cytoplasm of neighboring cells. Second, the propagation of tau aggregates was not accompanied by other pathological changes, such as neuronal loss, gliosis, inflammation, and axonal damage: The relationship between aggregate spreading and other degenerative changes has to be elucidated. Third, it needs to be addressed which molecular species are responsible for aggregate propagation and whether these species are identical to the species responsible for neurotoxicity. A related issue is whether the aggregate propagation mechanism involves co-factors cooperating with the aggregate itself. Resolution of these questions may open up opportunities for novel therapeutic and diagnostic strategies for major neurodegenerative diseases.

This is a provocative and exceptionally well-done study that convincingly demonstrates the CNS spread of tau pathology induced by the injection of P301S transgenic (tg) mouse brain homogenates containing P301S pathological mutant tau into the brains of wild-type tau tg and non-tg mice. However, I do think the use of the term “transmission” in the paper is unfortunate because this conjures up the idea that tauopathies may be infectious diseases like transmissible spongiform encephalitises (TSEs), as exemplified by mad cow disease and Creutzfeldt-Jacob disease (CJD). The concern comes from the fact that Alzheimer’s disease (AD) is the most common neurodegenerative tauopathy and this report could raise unwarranted worries on the part of the public or public health officials that AD is infectious and could be spread by contact with the millions of AD patients throughout the world. Although many studies by Carlton Gajdusek (e.g., see Godec et al., 1991) and others over the years failed to show reproducible evidence of transmission of AD by injecting AD brain homogenates along the lines in the Clavaguera et al. study, a report just over 20 years ago suggesting that this occurred (see Manuelidis et al., 1988) did precipitate undue public concern that AD might be transmissible/infectious. That said, the studies here show in a convincing manner that, following brain injection of P301S mutant tau pathological material, there is some form of cell-to-cell spread of pathological tau in the form of paired helical filaments (PHFs), which are amyloids just like prion, Aβ, and α-synuclein fibrils in CJD, AD, and Parkinson disease (PD), respectively. These PHFs are the building blocks of neurofibrillary tangles (NFTs), hallmark brain lesions of AD, as well as other tauopathies known as frontotemporal lobar degeneration (FTLD) of the tau type, or FTLD-tau, as exemplified by Pick disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and others (see MacKenzie et al., 2008). Thus, the findings here are important for understanding mechanisms underlying the onset as well as the progression of AD and related tauopathies.

I do not think the major implications of this paper are that there should be public health concerns that AD might be an infectious disease like CJD for the reasons discussed above. However, the paper has significant potential implications for how we think of disease pathogenesis for AD, other tauopathies, and other neurodegenerative brain amyloidoses like PiD, PSP, CBD, and PD. For example, can the model systems here explain the stereotypical manner in which the tau, Aβ, and α-synuclein amyloid pathologies appear to evolve (“spread”) over time with disease progression in most cases of these disorders? Obviously, more must be done to take this research further to explain these phenomena, but even the phenomena themselves can be used to argue against the cell-to-cell spread of each of these pathologies or combinations of them as exemplified by studies showing that the evolution and distribution of tau pathology in AD, PiD, CBD, and PSP are very different with different regions and brain cells being affected in different tauopathies. Indeed, NFTs occur commonly in both neurons and glia in some of these disorders, but mainly in neurons in other tauopathies despite the fact that the affected neurons are surrounded by glial cells which are unaffected by tau pathology. Endothelial cells in blood vessels that permeate brain profusely also do not show tau pathology in tauopathies. However, studies like the one here may provide strategies to explain these phenomena as well as the selective vulnerability of one group of neurons and glia to develop tau or other amyloid pathology compared to other groups of cells that appear resistant to accumulating tau pathology within the brain of a patient with AD, PSP, PiD, or CBD. These model systems also could address the issue of how/why patients with diseases like AD show other amyloid deposits, since α-synuclein amyloid deposits as Lewy bodies commonly occur in AD brains in addition to plaques and tangles, or why plaques and tangles occur in close proximity in some but not all affected brain regions in AD. To address this issue experimentally, we conducted studies similar to those in the current report, and we showed that injections of human AD PHFs into rat brains induces aggregation and deposition of Aβ (see Shin et al., 1993) and that tau and α-synuclein can induce each other to form amyloid fibrils in vivo and in vitro (see also Lee et al., 2004 and Giasson et al., 2003), yet the mechanisms of how this occurs and the significance of this are poorly understood. Further, AD patients also show evidence of accumulations of TDP-43 deposits, but TDP-43 pathology is neither congophilic nor shows features of amyloid, but it is characteristic of FTLD of the TDP-43 type or FTLD-TDP and ALS, and a recent study of AD patients demonstrated correlations between the presence of TDP-43 and α-synuclein pathologies and cognitive deficits in AD patients in addition to those attributable to plaques and tangles (see Nelson et al., 2008), so these additional TDP-43 and α-synuclein pathologies appear to be clinically relevant for AD.

These studies should be followed to address the types of issues mentioned above, and many others, and it would have been interesting if the authors had done studies to see if their P301S tg mouse brain injections induced Aβ and α-synuclein deposits, but that may be in the works already.